Ischemic heart disease and its associated complications of sudden cardiac death and congestive heart failure remain leading causes of morbidity and mortality in the United States. The arrhythmogenic pathophysiology of ischemic disease includes loss of cardiomyocyte electrical coupling through altered localization and modulation of gap junctions. The precise molecular mechanisms underlying altered coupling remain elusive. The objective of this proposal is to understand the cellular movement of gap junction hemichannels (connexons) in normal and ischemic conditions. Our central hypothesis is that connexons require the cytoskeleton to target them to specific locations on the plasma membrane and, once in the plasma membrane, there is a limited role of lateral diffusion and other means of non- cytoskeleton based channel movement. The hypothesis will be tested by using fixed and live cell imaging techniques including high resolution total internal reflection fluorescence (TIRF) imaging, with supplementary biochemistry, to understand the molecular mechanisms of connexon trafficking. Particular aims include understanding the role of microtubule based directed targeting of connexons to intercalated discs in conditions of oxidative stress and simulated ischemia; to understand the role of the actin cytoskeleton in targeted delivery of connexons; and to determine quantitatively the capacity of connexons to diffuse laterally to other membrane regions within the plasma membrane. Preliminary data indicate that oxidative stress limits microtubule capture of cortical membrane, preventing delivery of connexons to the plasma membrane; that actin helps microtubules position and target ion channels to specific regions of the cortical membrane, and that lateral diffusion of connexons is highly restricted. These aims will further our understanding of the regulation and behavior of cardiac gap junctions. The general field of protein trafficking will benefit from fundamental new knowledge about cytoskeleton and ischemic type regulation of these channels. Furthermore, key molecules and events in the trafficking of gap junctions will be identified to be used as therapeutic targets to lessen the arrhythmias and dysfunction associated with ischemic heart disease. PUBLIC HEALTH RELEVANCE: The clinical sequelae of ischemic heart disease are congestive heart failure and sudden cardiac death which are primary causes of mortality in the United States. The cellular basis of both heart failure and sudden death involve diminished electrical coupling between heart cells. This application proposes to study the molecular mechanisms of electrical coupling between heart cells and identify proteins involved in regulating the coupling under normal conditions and during times of reduced blood flow (ischemia).